For many decades, basic scientists considered asthmatic inflammation as a CD4+ T-helper II (Th2) cell response orchestrated by Th2-synthesized and Th2-secreted cytokines and growth factors. Consequently, many studies have centered around the role aberrant signaling of T helper cell subsets plays in the development of the eosinophilic inflammation typical of asthma. Recent reports, however, suggest roles for other T-cell subsets, the airway microenvironment, and the innate immune system. Although the eosinophil is the most numerous immune effector cell type that propagates chronic asthmatic inflammation, few signaling molecules have successfully been targeted to attenuate its inflammatory functions. The Rho-family of proteins, Cdc42 and Rac implicated in allergen-induced airway inflammation and hyper-reactivity, activate their main effectors serine/threonine p21-activated kinases (PAKs). Through actin-binding effectors, PAK1 modulates the actin cytoskeleton in mast cell degranulation, and additionally we have shown that this isoform is expressed in eosinophils. Eotaxin induced by the allergic sensitization and challenge process powerfully recruits eosinophils to the lungs via CCR3 signaling. We hypothesize that PAK1 modulates eotaxin-mediated eosinophil migration and infiltration and examine its effect using a Pak1 knockout (Pak1-/-) murine system. Previous work we have published and our preliminary data using this model suggest that differential blood cell counts as well as T helper subset function is preserved in the allergen-sensitized and challenged Pak1-/- mouse. Furthermore, eosinophil infiltration in the allergen-sensitized and challenged mouse as well as eotaxin-mediated eosinophil infiltration in vivo and chemotaxis in vitro are impaired by Pak1 genetic deletion. Here we show that PAK1 is activated by the eotaxin: CCR3 signaling in murine eosinophils. Two proposed aims will now focus on delineating the biochemical mechanism by which PAK1 modulates eotaxin-mediated eosinophil migration. In the first aim, we will target and restore PAK1 to thoroughly interrogate the role it plays in eotaxin-mediated migration and F-actin polymerization and depolymerization. Moreover, studies in our second aim will utilize lentiviral expression of PAK1 mutants to dissect out the specific mechanism by which PAK1 exerts this effect. We predict that the results in this project will inform the testing of anti-PAK therapy in preventing the development of eosinophilic inflammation in murine models of asthma.